The human diseases, the screening of the pathogenic genes and the functional research thereof are always important research fields. However, due to the systematic limitation, the possible living mechanisms are understood mostly through animal models which contain manipulated genes. For hundreds of years, Drosophila Melanogaster have been used as the basic source material for teaching and genetics study. In the last few decades, since the molecular biology method was brought into this field, the research of Drosophila molecular developmental genetics has entered into a new stage. The biological mechanism of Drosophila is very evolutionarily conserved. Therefore, from Drosophila study, people have come to understand that many human disease genes and cancer-related genes are derived from the gene mutation which controls the developmental mechanism. For example, the initiative mechanism of colon cancer and its complex working process are understood by studying the related genes of Wingless pathway which drives the development of abdominal segment formation in Drosophila embryo. Therefore, the main future studying direction of the present invention is doing the searching and studying of the specific functional genes in Drosophila genome based on the technique of the present invention. The Drosophila model will then be the base of searching the homologous genes of the human disease and pathogenic genes for the further study of human genetic mechanism.
In the present time, as the structural genomic projects in model organisms are completed, how to decipher a large amount of raw DNA sequences data in understanding gene function in vivo will be one of the major tasks for biology-related researchers. Different genomic strategies for defining and dissecting developmental and physiological pathways have been approached. The summit of these approaches is the systematic genomic screening of a specific functional trait using DNA tags such as P-transposon or retrovirus as mutagenesis agents. The designed transposon can be used as a mobile element to rapidly obtain cellular DNA sequence nearby the genetic mutation. The cellular DNA sequences can be obtained from the P-transposon-induced mutated genes loci by the direct use of inverse PCR (IPCR) or plasmid rescue methods. By such operating way, the inefficient and labor-intensive drill of cloning sequences for obtaining the junction sequence between the host and the mobile element can be circumvented. While retrovirus was used to mutate leukemia-causing genes in mouse (Li et al., 1999, Nature Genetics 23, 348), several hundred integration sites were cloned and characterized followed by high-throughput sequencing, data analysis and refined genetic mapping. Similarly, a global transposon mutagenesis in Mycoplasma allowed the question of the number of essential genes in a minimal genome to be answered (Hutchison, et al., 1999, Science 286, 2165). Therefore, by combining with emerging genomic tools, such systems in model organisms will indeed dramatically accelerate the pace of discovery in human disease-genes and cancer-related genes.
For the functional study of Drosophila genes, a conventional approach relies on the creation of mosaic animals whereby the genotype varies in a cell-specific or tissue-specific manner. Currently, various techniques utilize the yeast FLP-FRT recombination system introduced into Drosophila (Golic and Lindquist, 1989, Cell 59, 499) to promote chromosomal site-specific exchange. This system allows the efficient recovery of homozygous patches in an otherwise heterozygous animal and thus permits a phenotypic analysis of mutant tissues.
Different versions of the FLP-FRT (Fippase-Flippase Recombination Target sequence) system have been established for analyzing gene functions in either somatic or germline tissues. The direct mosaic productions in different somatic tissues have been established (Xu and Rubin, 1993, Development 117, 1223; Duffy et al., 1998, Development 125, 2263). In these methods, different tracing markers are used as the controls to monitor the presence of homozygous clones of genes to be studied. In addition, the FLP-DFS technique suitable for asking germline functions for loci residing in more than 95% of the genome has also been systematically completed (Chou and Perrimon, 1992, Genetics 131, 643; Chou et al., 1993, Development, 119, 1359 and Chou and Perrimon, 1996, Genetics 144, 1673). The FLP-DFS technique uses the X-linked germline-dependent dominant female sterile mutation ovoDI as a selection marker for the detection of germline recombination events. Nevertheless, the FLP-FRT system is used to promote site-specific chromosomal exchange (Chou and Perrimon, 1992, Genetics 131, 643).
However, the major drawback for all of these FLP-FRT methods is that the mobile element such as the P-transposon can not be used directly as the mutagenesis agent to mutate the FRT chromosomes. While Δ2-3 transposase is recognizing the P transposon insertion as the mobilization origin, it simultaneously recognizes and transposes the P[FRT] insertions used in the FLP-FRT system. Under such situation, the genetic recombination cannot proceed due to the fact that the P[FRT] chromosomes are not homologous. For example, the mobilized P[FRT] will mostly create a non-homologous condition. However, the germline recombination of P[FRT]-ovoDI chromosome needs the existence of the homologous P[FRT] chromosome when P[FRT]-ovoDI chromosome is used for the FLP-DFS germline recombination. Therefore, the transposition of the P[FRT] insertion results in a non-homologous condition so that the germline recombination cannot proceed.
Presently, only EMS-based methods can be used for a full-scale genome-wide screening when using FRT chromosomes. Many interesting genes have been recovered. However, the goal to completely recover and to do molecular characterization of all interesting loci efficiently would be difficult if only EMS-based methods are used for mutagenesis. Because EMS produces mostly point mutations, it does not create any molecular tags on mutated genes for cloning manipulation. Consequently, the approach for identifying important genes is heavily impeded by the inefficient and labor-intensive traditional molecular cloning procedures.
Another alternative strategy to facilitate gene cloning is to use transposition system independent of the P transposon. For example, the Hobo element system can be used to cause the gene mutation. However, the problem of creating a chromosomal environment that completely avoids the P transposon system has not been possible in the Drosophila field. This kind of approach has never been described in the field while the versatile FLP-FRT system has been publicized since 1989 (Golic and Lindquist, 1989, Cell 59,499).
Another way to overcome this is to individually recover the recombinant chromosome with an interested P insertion and the specific P[FRT]. This tedious and laborious work has been done for a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project (Perrimon et al., 1996, Genetics 121, 333). By using the FLP-DFS technique, 496 independent zygotic lethal mutations identified by single P-element mutations were tediously recombined with FRT chromosomes in order to analyze their germ-line clone phenotypes (Perrimon et al., 1996, Genetics 121, 333). Similarly, the same approach has been conducted in at least 7 labs in Europe. They collected only 700 recombinant chromosomes within at least 6 years. The strategy to perform similar recombination experiment is confronted with not only the prerequisite to know the location of the new P insertion for a successful recombinant but also the limitation of reaching a saturation screening since the recombination suppression exists in certain chromosome regions. These recombinants are too few to reach the 24,300 lethal chromosomes in order to reach a 87% saturation screening for the functional description of Drosophila essential genes (Spradling, 1999, Genetics 153, 135).
In order to overcome the foresaid drawbacks, the present invention circumvents the above difficulties by constructing an advanced version of P[FRT] insertions on the Drosophila second chromosome, which allows the P-directed mutagenesis to be useful for quick chromosome-wide screening and fast molecular cloning for the various FLP-FRT methods. Molecular biology technique such as inversed PCR (polymerase chain reaction) and plasmid rescue methods can be used to recover flanking genomic DNA sequences and relevant molecular properties of the genes affected by the transposon. Based on the mutated phenotypes of either germline or somatic recombinant clones produced, the biological functions can be described for the genes mutated. The integrated description of the molecular nature and biological function of Drosophila genes can accelerate the understanding of the functions of human gene homologues and be used as the basis for the application and development of gene-based medicines.